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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.08.-9.11.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
(see Overall remarks)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Name of test material (as cited in study report): Solvent Yellow 124
Substance type: organic
Physical state: liquid
Appearance: dark yellow viscous liquid

Composition of test material, percentage of components:
- Analytical purity: 90.0 % (w/w)
- Impurities (identity and concentrations):
4´-[2-((hydroxy)ethyl)ethylamino]azobenzene 3.0 % (w/w)
1,1-bis(N-ethyl[4-(phenylazo)phenyl]aminoethan-2-oxy)ethan 2.5 % (w/w)
- Unknown impurities 4.5 % (w/w)

Lot/batch No.: S2408
Expiration date of the lot/batch: Unlisted
Stability under test conditions: stable
Storage condition of test material: During the study the test substance was stored in glass bottle at laboratory temperature.

Method

Target gene:
gene for synthesis histidine or tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requiring strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan requiring strain
Metabolic activation:
with and without
Metabolic activation system:
postmitochondrial fraction (S9)
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide for analysis (purity>99.9%), Merck, Lot No. K41063052 028
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
(Dimethylsulfoxide)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylenediamine; 9-aminoacridine hydrochloride monohydrate; 2-aminofluorene; 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation)

NUMBER OF REPLICATIONS:
two series of experiments were performed with each strain - without and with metabolic activation, triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the aplication of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods (2, 3).
2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no toxicity with TA 100 (without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
ambiguous slight mutagenic effect
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The results obtained in most experiments did not show substantial (biologically significant) increases in the number of revertants versus solvent controls (Rt/ Rc < 2), and no experiment gave evidence of a rising trend in the number of revertants with increasing dose.

In some cases, experiments with increased values of revertants occurred, exclusively with metabolic activation. It concerns the following experiments:

- TA 100 +MAII, trend to 500 µg per plate Rt/Rc max =1.5;

- TA 1535 +MAI trend to 1500 µg per plate Rt/Rc max =1.7;

- TA 98 +MAI trend to 500 µg per plate ,max Rt/Rc 1.9 and experiment +MAII, max  Rt/Rc =1.6;

- E. coli + MAII, without any trend, max. Rt/Rc =1.5; increase caused rather by lower solvent control value.

 

In case of TA 100, TA 1535 and E.coli the results were neither confirmed by the parallel experiments nor Rt/Rt reached 2. In case of TA 98, increased values of revertants were observed in both experiments, while dose-response relationship was observed in the first experiment only. It could be caused relatively small enhancement of number of revertants and large variability of the biological system. Anyway, such increasing is not common in negative experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
other: negative, but result of TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect

Under the above-described experimental design, the test substance Solvent Yellow 124 was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains in experiments without metabolic activation. It was nonmutagenic also for Salmonella typhimurium TA 100, TA 1535 and TA 1537 and for Escherichia coli WP2 uvrA with metabolic activation. The results in Salmonella typhimurium TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect.
Executive summary:

Test substance Solvent Yellow 124 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test.

The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 5-5000 µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Solvent Yellow 124 was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains in experiments without metabolic activation. It was nonmutagenic also for Salmonella typhimurium TA 100, TA 1535 and TA 1537 and for Escherichia coli WP2 uvrA with metabolic activation.

The results in Salmonella typhimurium TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect.